Pest-sequencing analysis of periodontal pathogen Porphyromonas gingivalis : expression of its proteins by oral environmental fectors.

牙龈卟啉单胞菌是一种专性厌氧菌，与慢性牙周炎的发生发展密切相关。虽然已经发现细菌的几个毒力因子的表达受到环境胁迫的影响，如进入生长稳定期和高温，但关于细菌对环境胁迫的反应机制的信息相对较少。在这项研究中，我们研究了在二维凝胶分析后初步鉴定的新蛋白(USTA)。在稳定期或暴露于大气氧气中，USTA的表达上调。N-末端测序和数据库分析表明，USTA编码基因(USTA)位于USP基因的上游，USP基因编码普遍的应激蛋白。引物延伸和Northern印迹分析表明，USTA基因以单顺反子方式转录。为了阐明USTA在细菌中的作用，构建了携带USTA基因中断的染色体突变体。突变株在富营养液中的生长速度慢于野生型亲本菌株，导致稳定期产量较低。此外，在该突变体中，牙龈假单胞菌超氧化物歧化酶、硫醇过氧化物酶和硫氧还蛋白同源物的表达水平明显高于野生型，尤其是在稳定期。与野生型相比，Usta突变体对联胺的抗性更强。联胺是一种硫醇特有的氧化剂。此外，USTA突变抑制了oxR突变体对联胺、甲硝唑和丝裂霉素C的超敏反应。这些结果表明，USTA可能在细菌的氧化应激反应中发挥重要作用。

Porphyromonas gingivals, an obligate anaerobic bacterium, is implicated as a major pathogen in the development and progression of chronic periodontitis. Although expression of several virulence factors of the bacterium has been found to be affected by environmental stress such as entrance into the stationary growth phase and heat, there is relatively little information on the mechanisms that may operate in the bacterium in response to environmental stress. In this study, we investigated the new protein (UstA) that was initially identified following the two dimensional gel analysis. Expression of UstA was upregulated in stationary phase or by exposure to atmospheric oxygen. N-terminal sequencing and database analysis with the P.gingivalis genome sequence revealed that the UstA-encoding gene (ustA) was located upstream of a homologue of the usp gene encoding the universal stress protein on the chromosome. The ustA gene appeared to be transcribed in a monocistronic fashion as revealed by primer extension and Northern blot analysis. To elucidate the role of UstA in the bacterium, chromosomal mutants carrying a disruption of the ustA gene were constructed. The ustA mutant grew slower than the wild type parent strain in rich medium, resulting in a lower yield in stationary phase. Furthermore, in this mutant, the expression levels of the P.gingivalis homologues of superoxide dismutase, thiol peroxidase, and thioredoxin were markedly higher than those in the wild type especially in stationary phase. The ustA mutant was more resistant to diamide, a thiol-specific oxidant, than the wild type. In addition, the ustA mutation suppressed hypersensitivities of the oxyR mutant to diamide, metronidazole, and mitomycin C. These results suggest that UstA may play a significant role in oxidative stress responses in the bacterium.